The objective of this proposal is to use planarians as a metazoan model system in which to dissect and manipulate the molecular basis of animal regeneration. Planarians have been chosen as a model system because of their key position in metazoan phylogeny, their remarkable regeneration abilities, and the totipotential nature of these animals' regenerative stem cell population (neoblasts). First, we propose to identify and characterize the genes involved in specifying and maintaining anterior and posterior regeneration fates. We will accomplish this objective using a polymerase chain reaction-based gene-screen. The methodology involves the subtractive hybridization of cDNAs from regenerating and non-regenerating tissues, and then amplifying the enriched cDNAs unique to each tissue (see preliminary results). The gene-screen-isolated cDNAs will be characterized by sequencing, developmental Northerns and in situ hybridizations. Secondly, we will take advantage of the asexual and sexual reproductive strategies of planarians, and of their totipotential regenerative stem cells to develop the methodologies necessary for the production of transgenic animals. Transgenesis will allow functional testing of those genes whose sequence, developmental expression profile, and tissue specificity indicate potential regulatory roles during the process of regeneration. Once identified, the functional characterization of regeneration-specific genes in planarians will be vertically integrated to the study of regeneration in higher organisms. Ultimately, the genes identified in planarians, and their interactions during regeneration will define a series of useful molecular templates which will help unravel the more complex epigenetic processes of vertebrate regeneration, and uncover the factors which make regeneration permissive in some, but not all metazoans.